Each of biopolymers has specific biological active property. Therefore, they are expected to be used as elemental materials for regenerative medicine and drug delivery systems. However, many biopolymers themselves are water soluble at physiological temperatures. Therefore, in order to use biopolymers for medicine, they are chemically modified with a low molecular compound or are crosslinked. In general, chemical modification of biopolymers is carried out by a method involving a chemical reaction in a solution for forming/processing or a method wherein a preliminarily prepared structure is immersed in a solution containing a compound (e.g., a crosslinking agent) that can cause chemical modification.
Meanwhile, in recent years, electrospinning has been gaining attention as a technique for readily producing submicron-scale fibers. In the case of such technique, fibers are formed by injecting a polymer solution while applying a voltage to the solution. The fiber thickness depends on applied voltage, solution concentration, and the distances that sprayed particles move. A thin film having a three-dimensional structure (three-dimensional mesh structure) can be obtained by continuously forming fibers on a substrate. In addition, a film having a fabric-like thickness can be produced by the aforementioned method, and a non-woven fabric having a submicron-scale mesh structure can be produced. Applied use of such non-woven fabric for spacesuits and protective suits is being studied. The above technique is used for formation of a structure used in the field of medicine with the use of biopolymers (JP Patent Publication (Kohyo) No. 2004-532802 A and JP Patent Publication (Kokai) No. 2004-321484 A).
JP Patent Publication (Kohyo) No. 2004-532802 A describes that a collagen structure which was preliminarily prepared by electrospinning is exposed to a glutaraldehyde vapor so that the structure is crosslinked. However, such technique involves crosslinking merely caused by exposing a collagen structure to a volatile glutaraldehyde gas. Glutaraldehyde has been widely used as a crosslinking agent for collagen and gelatin. However, glutaraldehyde itself is toxic. In addition, a glutaraldehyde-derived structure is introduced in the obtained crosslinked structure. Therefore, there is a possibility to lose useful characteristics inherent in collagen or gelatin. In addition, there is a possibility that the structure could become unexpectedly toxic.
Meanwhile, there is a method for crosslinking collagen and gelatin with an enzyme. A known enzyme used for crosslinking collagen and gelatin is transglutaminase. Transglutaminase is an enzyme that allows glutamine residues and lysine residues to bond with each other. In general, an enzyme exhibits activity specifically at an optimum temperature, and it exhibits significantly lowered activity at a non-optimum temperature (e.g., Activa TG-S, produced by Ajinomoto Co., Inc. with an optimum temperature of approximately 50° C.). Crosslinking of gelatin with transglutaminase normally takes place at 30° C. to 60° C., at which gelatin can be dissolved in water. Therefore, when crosslinking is caused with the use of transglutaminase without dissolution of a structure comprising gelatin, it is necessary to cause a reaction at 20° C. or less at which gelatin is not dissolved in water (JP Patent No. 3012743). However, when a structure comprising gelatin is immersed in water at a low temperature, a structure might become significantly deformed, which is problematic. In such case, it would be difficult to cause efficient crosslinking while preventing deformation of a preliminarily prepared gelatin structure.
In addition, biopolymers, which are biologically derived polymers, have specific physiological and biological properties and therefore they are frequently used for medical and biological purposes. However, since biopolymers are generally water soluble, they lack the physical strength that is necessary for chemical modification or crosslinking when used in a solid form. In view of the above, biopolymers are coated to synthetic polymers and inorganic substances (Brash, Trans. Am. Soc. Artif. Int. Organs, p. 69, 1974), or hybrid products (composites) are formed with such materials and biopolymers. However, in consideration of biological safety, materials consisting of biopolymers only are preferably used.
For improvement of biodegradability and strength of a biopolymer and water insolubility of a biopolymer, a technique of chemically modifying a partial functional group of a biopolymer and a technique of crosslinking between biopolymers have been devised. In particular, crosslinking between biopolymers has been actively studied. For instance, crosslinking of gelatin with the use of glutaraldehyde or a condensation agent has been widely carried out. It is reported that this method is effective in the field of regenerative medicine and in the drug delivery field (JP Patent No. 3639593). However, the degree of crosslinking and the strength achieved by such crosslinking are insufficient. Therefore, applied use of biopolymers for medical purposes is limited.
For crosslinking of gelatin, a method for crosslinking gelatin with glutaraldehyde is generally used. In this method, it is necessary to carry out stirring at a temperature at which gelatin can be dissolved (i.e., 30° C. or more). However, the reactivity of gelatin and glutaraldehyde becomes high at such temperature. As a result, it becomes impossible to uniformly stir the entire solution. In such case it is impossible to use a crosslinking agent at a high concentration. Thus, it has been difficult to produce gelatin having a high degree of crosslinking. Accordingly, it has been difficult to produce gelatin having a high strength and a high degree of crosslinking.
JP Patent Publication (Kohyo) No. 2002-531182 A describes that a matrix for tissue construction that comprises a protein is produced by dissolving the protein and alkyl ester of hyaluronate in HFIP, followed by forming. In addition, JP Patent Publication (Kohyo) No. 2004-532802 A and JP Patent Publication (Kokai) No. 2004-321484 A describe that a protein structure is produced by electrospinning. However, in these methods, crosslinking is not carried out in HFIP. In addition, there is an example involving crosslinking of fibers with the use of glutaraldehyde, such fibers being produced by electrospinning using HFIP as a solvent (Biomaterials, 27(8), 452-1461, 2006). However, this method merely involves crosslinking of a prepared structure with the use of a glutaraldehyde vapor.